A large number of mitochondrial proteins are subject to reversible lysine acetylation, suggesting that this modification plays a significant role in mitochondrial biology. Three distinct NAD+dependent protein deacetylases, called SIRT3, 4 and 5, have been identified in mitochondria. The PIs of this proposal have extensively collaborated to characterize the phenotype of knockout mice lacking SIRT3. These mice show marked mitochondrial protein acetylation. SIRT3 expression increases during fasting and activates (3-oxidation of fatty acid in the liver. SIRTS directly deacetylates two key mitochondrial enzymes in the fatty acid oxidation pathway and increases their enzymatic activities. Metabolomic analysis of mice lacking SIRTS shows significantly greater levels of acylcarnitines during fasting than wild-type mice. SIRTS-/- mice also show reduced ATP levels and intolerance to cold exposure upon fasting consistent with their fatty acid oxidation defect. When SIRTS-/- mice are placed on a high fat diet, they show accelerated development of a syndrome that closely mimics the human metabolic syndrome: obesity, type II diabetes, lipid abnormalities, steatohepatltis and hepatocellular carcinoma. These findings identify acetylation as a novel regulatory mechanism for mitochondrial metabolism. This proposal will use knockout and transgenic mice for SIRTS, 4 and 5 to further explore the mitochondrial acetylproteome, i.e. the proteins that are reversibly deacetylated by SIRTS, 4 and 5. The metabolome of mice lacking SIRTS, 4 and 5 will be explored in a variety of organs to identify metabolic pathways that are regulated by individual mitochondrial sirtuins. We will further explore the molecular mechanism(s) responsible for the development of type II diabetes in mice lacking SIRTS and its link to disordered lipid metabolism. Finally, we will study the regulation of expression of mitochondrial sirtuins under different physiological conditions and screen a small molecule library for drugs that enhance SIRTS expression. The effect of these drugs on the manifestations ofthe metabolic syndrome will be examined in mice. We anticipate that these experiments will increase our understanding of the role of reversible mitochondrial protein acetylation and its enzymes in metabolic regulation and in type II diabetes. PUBLIC HEALTH RELEVANCE: Mitochondria are key players in the pathogenesis of several metabolic disorders. The recent identification of reversible lysine acetylation on a large number of mitochondrial proteins and the identification of three mitochondrial sirtuin (SIRTS, 4 and 5) suggest that reversible mitochondrial acetylation plays a role in the pathogenesis of metabolic disorders. Understanding the role of SIRT 3, 4, 5 in the pathogenesis of the metabolic syndrome and other metabolic disorders could yield novel therapeutic opportunities.